Oxidative stress is an important component of toxicity due to environmental, occupational and therapeutic agents. Two major thiol-containing defense systems exist to protect against oxidative stress, one utilizing the tripeptide glutathione (GSH) and the other using a small protein, thioredoxin (Trx). These systems have similar NADPH-dependent reductases and have overlapping functions and activities. However, there are also important differences, such as the role of thioredoxin in maintaining transcription factors in their reduced and functional forms. Although a considerable amount of information exists on the redox responses of the GSH system during redox signaling and oxidative stress, little information is available on the redox of Trx. The purpose of this proposal is to use a novel Redox-Western blot technique to investigate the redox of Trx in cells, and use this in combination with cell fractionation to measure redox of the nuclear and cytoplasmic Trx pools. Redox of cellular GSH/GSSG and NADPH/NADP+ will be simultaneously determined to gain insight into the control and interaction of these redox pools. The first aim is to determine the redox state of nuclear and cytoplasmic Trx in HT29 cells during cell growth, differentiation and apoptosis. The second aim is to determine whether nuclear translocation of Nrf2, a transcription factor activated by oxidative stress and implicated in regulation of detoxification gene expression, is specifically associated with oxidation of cytoplasmic Trx redox while nuclear Trx remains reduced. The third aim is to selectively modify Trx redox in nuclear and cytoplasmic compartments to examine cause-effect relationships between Trx redox and transcriptional activation. This will be done with retroviral constructs containing mitochondrial Trx reductase in which the mitochondrial targeting sequence has been removed and a nuclear import or export signal has been added. Successful completion of these aims will quantitatively define the redox of nuclear and cytoplasmic Trx, show whether Trx and GSH have distinct functions in cell signaling and oxidative stress, and test whether nuclear or cytoplasmic Trx redox is critical in transcriptional activation during oxidant signaling.